Increasing awareness has been placed in understanding the issues related to rehabilitation of the estimated 330,000 service personnel diagnosed with traumatic brain injury (TBI) since the start of Operation Enduring Freedom and Operation Iraqi Freedom, who suffer from physical, cognitive, emotional dysfunction and recurrent pain. Specifically, opioid misuse is a growing epidemic nationwide, and recent evidence indicates that Veterans who sustain moderate/severe TBI are more likely to receive long-term treatment with opioid- based medications and engage in higher-risk opioid use patterns. These findings highlight the critical need for studies to establish what adaptive changes occur in the opioid system in the injured brain that may influence nociceptive, cognitive and reward/dependence outcomes. Morphine, the prototypical opioid antinociceptive, binds to the mu opioid receptor (MOR) to stimulate G-protein signaling and downstream effectors, such as extracellular signal-regulated kinase (ERK). In addition to this classical signaling pathway, morphine can act through the immunity toll-like like receptor 4 (TLR4) to activate glial cells and stimulate the release of proinflammatory mediators, like interleukin 1 beta (IL-?1). While both signaling pathways contribute to morphine-induced phenotypes, evidence indicates immune activation disrupts morphine?s efficacy to promote analgesia, worsens pathological pain and exacerbates dependence and withdrawal. While TBI is recognized to induce long-term effects on immune system function alone, to date, no published papers have explored the combined influence of TBI and opioids on these signaling outcomes, adding to the significance and innovation of the proposed project. The current SPiRE proposal will test the hypothesis that repeated exposure to morphine following TBI will upregulate MOR expression/function and selectively potentiate glial-mediated inflammatory processes upon subsequent re-exposure to morphine in regions associated with reward, cognition and pain. The objectives in this proposal will be accomplished through two Specific Aims with tissue measurements in the nucleus accumbens, ventral tegmental area, prefrontal cortex, periaqueductal gray, dorsal hippocampus and amygdala. Aim 1 will assess longitudinal changes in MOR status as measured by radioligand binding density/affinity as well as gene expression (real-time PCR) and immunofluorescent co- localization on neurons and glia in tissue collected 7 days, 1, 3 and 6 months following TBI/repeated morphine. Aim 2 will characterize differential activation of classical and inflammatory pathways stimulated by morphine re- exposure at long-term timepoints (3 and 6 months) following combined TBI and subchronic morphine. Activity through classical pathways will be evaluated by G-protein (GTP?S) activity and extent of phosphorylated ERK immunofluorescence. Inflammatory pathway signaling will be assessed by measuring the extent of glial activation with immunofluorescence, TLR4 microglial staining and flow cytometry, and IL-?1 protein quantification with ELISA. The hypothesized finding of a potentiated inflammatory response to morphine following TBI would be consistent with conferring an increased risk of adverse outcomes associated with activation of this pathway, including addiction vulnerability, impaired cognition and hyperalgesia, making these studies highly relevant to the mission for VA patient care and rehabilitation. These data will also provide critical evidence to serve as a foundation for a comprehensive future study to explore the behavioral implications of these signaling outcomes and pharmacologic strategies exploring biased agonism.